Multiplexed volumetric bar chart chip for point of care biomarker and analyte quantitation

ABSTRACT

Apparatus for determining the quantity of a target protein, biomarker or analyte present in a sample, comprising a top plate and a bottom plate, each comprising a plurality of recesses arranged to form a plurality of rows extending parallel to one another, and a plurality of channels extending perpendicularly to the plurality of rows of the bottom plate; wherein the top plate and the bottom plate are assembled together so that the top plate is on top of the bottom plate and the recesses of the top plate communicate with the recesses of the bottom plate so as to form a plurality of rows; and wherein at least one of the top and bottom plate is configured to slide relative to the other of the top and bottom plate in order to form a plurality of columns, with each of the columns in communication with each of the channels.

REFERENCE TO PENDING PRIOR PATENT APPLICATION

This patent application claims benefit of pending prior U.S. ProvisionalPatent Application Ser. No. 61/714,676, filed Oct. 16, 2012 by LidongQin et al. for MULTIPLEXED VOLUMETRIC BAR CHART CHIP FOR POINT OF CAREBIOMARKER QUANTITATION (Attorney's Docket No. METHODIST-4 PROV), whichpatent application is hereby incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to methods and apparatus fordetermining the quantity of a protein and other biomarkers and analytespresent in a sample, and more particularly to methods and apparatus forpoint of care determination of the quantity of a protein (and,preferably, the quantity of multiple biomarkers) present in a sample.

BACKGROUND OF THE INVENTION

Molecular quantity analysis is widely used in research, diagnosis,quality control and other types of measurements. It is well known thatthe diagnosis and treatment of certain medical conditions can befacilitated by identifying the presence and quantity of a selectedbiomarker in a sample taken from a patient. Furthermore, research hasshown that, in many situations, multi-biomarker measurements can providea more accurate diagnostic result. More particularly, biomarker researchhas identified many helpful proteomics and genomic panels for diseasediagnosis and prognosis, including cancer, infection, cardiovasculardisease, diabetes, Alzheimer's disease and others. For example, afour-biomarker panel has been developed for detecting early stageovarian cancer, and an 18-protein biomarker panel has been developed forthe diagnosis of early Alzheimer's disease.

Current methods for protein-based biomarker assays typically utilize anenzyme-linked immunosorbent assay (ELISA) approach, where the targetprotein binds to a specific recognition molecule, and then colorimetric,fluorescent, electrochemical or magnetic signals are introduced totransduce the binding event into a readout signal. However, inasmuch asadvanced instrumentation is typically required for quantitativedetection of the target protein, these methods are not ideal for pointof care applications, due to the size and high cost of theinstrumentation and/or the complicated operation of the instrumentation.See, for example, FIG. 1, which shows the typical approach for aprotein-based biomarker assay, where a blood sample is drawn from apatient and then processed by a relatively large, complex instrument.

Thus there is a need for a new method and apparatus for point of caredetermination of the quantity of a protein (and, preferably, thequantity of multiple proteins) present in a sample.

SUMMARY OF THE INVENTION

These and other objects are addressed by the provision and use of anovel method and apparatus for point of care determination of thequantity of a protein (and, preferably, the quantity of multipleproteins) present in a sample.

In one form of the present invention, there is provided apparatus fordetermining the quantity of a target protein and other types ofbiomarkers or analytes present in a sample, the apparatus comprising:

a top plate comprising a plurality of recesses arranged to form aplurality of rows extending parallel to one another; and

a bottom plate comprising a plurality of recesses arranged to form aplurality of rows extending parallel to one another, and a plurality ofchannels extending perpendicularly to the plurality of rows of thebottom plate;

wherein the top plate and the bottom plate are assembled together sothat the top plate is on top of the bottom plate and the recesses of thetop plate communicate with the recesses of the bottom plate so as toform a plurality of rows; and

wherein at least one of the top plate and the bottom plate is configuredto slide relative to the other of the top plate and the bottom plate inorder to form a plurality of columns, with each of the plurality ofcolumns in communication with each of the plurality of channels.

In another form of the present invention, there is provided a method fordetermining the quantity of a target protein and other types ofbiomarkers or analytes present in a sample, the method comprising:

providing apparatus comprising:

-   -   a top plate comprising a plurality of recesses arranged to form        a plurality of rows extending parallel to one another; and    -   a bottom plate comprising a plurality of recesses arranged to        form a plurality of rows extending parallel to one another, and        a plurality of channels extending perpendicularly to the        plurality of rows of the bottom plate;    -   wherein the top plate and the bottom plate are assembled        together so that the top plate is on top of the bottom plate and        the recesses of the top plate communicate with the recesses of        the bottom plate so as to form a plurality of rows; and    -   wherein at least one of the top plate and the bottom plate is        configured to slide relative to the other of the top plate and        the bottom plate in order to form a plurality of columns, with        each of the plurality of columns in communication with each of        the plurality of channels;

binding a protein-specific antibody in at least one recess forming oneof the plurality of rows of the top plate;

positioning hydrogen peroxide in a recess adjacent to the row containingthe protein-specific antibody;

positioning ink in a recess in a row adjacent to the plurality ofchannels;

positioning a sample in the at least one recess containing theprotein-specific antibody;

positioning a catalase in the at least one recess containing theprotein-specific antibody and the sample;

sliding one of the top plate and the bottom plate relative to the otherof the top plate and the bottom plate so as to form the plurality ofcolumns, with each column being in communication with one of theplurality of channels; and

determining the quantity of the target protein and other biomarker andother molecular analyte present in the sample by detecting thelongitudinal position of the ink contained in the plurality of channels.

In another form of the present invention, there is provided a method fordetermining the quantity of a target analyte present in a sample, themethod comprising:

providing apparatus comprising:

-   -   a top plate comprising a plurality of recesses arranged to form        a plurality of rows extending parallel to one another; and    -   a bottom plate comprising a plurality of recesses arranged to        form a plurality of rows extending parallel to one another, and        a plurality of channels extending perpendicularly to the        plurality of rows of the bottom plate;    -   wherein the top plate and the bottom plate are assembled        together so that the top plate is on top of the bottom plate and        the recesses of the top plate communicate with the recesses of        the bottom plate so as to form a plurality of rows; and    -   wherein at least one of the top plate and the bottom plate is        configured to slide relative to the other of the top plate and        the bottom plate in order to form a plurality of columns, with        each of the plurality of columns in communication with each of        the plurality of channels;

binding a capture agent in at least one recess forming one of theplurality of rows of the top plate, introducing a sample into the atleast one recess so that an analyte contained in the sample is bound tothe capture agent, and binding a probe to the bound analyte; andpositioning a reagent in a recess adjacent to the row containing thecapture agent, bound analyte and bound probe; and positioning ink in arecess in a row adjacent to the plurality of channels;

sliding one of the top plate and the bottom plate relative to the otherof the top plate and the bottom plate so as to form the plurality ofcolumns, with each column being in communication with one of theplurality of channels; and

determining the quantity of the analyte present in the sample bydetecting the longitudinal position of the ink contained in theplurality of channels.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects and features of the present invention will bemore fully disclosed or rendered obvious by the following detaileddescription of the invention, which is to be considered together withthe accompanying drawings wherein like numbers refer to like parts, andfurther wherein:

FIG. 1 which shows a typical prior art approach for a protein-basedbiomarker assay, where a blood sample is drawn from a patient and thenprocessed by a relatively large, complex instrument;

FIG. 2 shows the novel multiplexed volumetric bar chart chip of thepresent invention;

FIG. 3 shows the novel multiplexed volumetric bar chart chip of FIG. 2and a barcode scanner which can be used to read the multiplexedvolumetric bar chart chip;

FIGS. 4-8 illustrate further details of the novel multiplexed volumetricbar chart chip of the present invention;

FIG. 9 is a schematic drawing of an etching process which can beutilized to form recesses and channels in the top plate and the bottomplate of multiplexed volumetric bar chart chip;

FIG. 10 is a schematic drawing of the assembly and operation of themultiplexed volumetric bar chart chip of the present invention;

FIGS. 11 and 12 are schematic drawings illustrating use of themultiplexed volumetric bar chart chip of the present invention;

FIG. 13 shows the multiplexed volumetric bar chart chip of the presentinvention prior to the oblique sliding of the top plate relative to thebottom plate;

FIGS. 14-16 show the test results obtained in accordance with thepresent invention for various samples;

FIGS. 17-20 show specific steps which are performed in accordance withthe method of the present invention;

FIGS. 21-32 are a schematic series of views illustrating the assemblyand operation of the multiplexed volumetric bar chart chip in one formof the present invention;

FIGS. 33-45 are a schematic series of views showing how, over time, theink in various bar channels advance in the multiplexed volumetric barchart chip according to the quantity of target proteins or other typesof biomarkers or other molecular analytes present in the sample;

FIG. 46 illustrates specific steps which are performed in accordancewith a DNA assay scheme and oxygen generation mechanism;

FIG. 47 shows an alternative embodiment of the novel multiplexedvolumetric bar chart chip of the present invention;

FIG. 48 shows images of hydrogen peroxide solution pushed into platinumwells using the multiplexed volumetric bar chart chip of FIG. 47;

FIGS. 49 and 50 show an alternative embodiment of the novel multiplexedvolumetric bar chart chip of the present invention; and

FIG. 51 shows an alternative embodiment of the novel multiplexedvolumetric bar chart chip of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new method and apparatus for point ofcare determination of the quantity of a protein (and, preferably, thequantity of multiple proteins) present in a sample.

More particularly, and looking now at FIG. 2, in one preferred form ofthe invention, there is provided a novel multiplexed volumetric barchart chip 5. Multiplexed volumetric bar chart chip 5 is configured tosimultaneously determine the quantity of multiple proteins which may bepresent in a sample, with the quantity of each protein which is presentin the sample being indicated in a particular one of a plurality of barchannels 10. By way of example but not limitation, 6, 10, 30, and50-plexed, or more than 50-plexed, channels may be incorporated intomultiplexed volumetric bar chart chip 5. Bar channels 10 may be straight(as shown in FIG. 2) or curved (e.g., serpentine, circular, z-shaped) orformed in any other configuration which provides a series of channelshaving a length. As a result of this construction, the review of aparticular bar channel 10 will indicate the quantity of a particularprotein which may be present in the sample and, significantly, thecollective array of the plurality of bar channels 10 will simultaneouslyindicate, in bar chart form, the quantities of multiple proteins whichmay be present in the sample, whereby to provide multi-protein quantitymeasurements and hence a more comprehensive diagnostic result.

As seen in FIG. 3, the multi-protein measurements presented in bar chartform by multiplexed volumetric bar chart chip 5 may then be read with asmart-phone or barcode scanner 15, whereby to automate the datacollection process.

Looking now at FIGS. 4-8, multiplexed volumetric bar chart chip 5comprises two plates, a transparent top plate 20 and a bottom plate 25(which may or may not be transparent).

Top plate 20 (FIGS. 5 and 6) has a plurality of recesses 30 formed onits bottom surface, with recesses 30 being arranged in a plurality ofrows 35 (i.e., 35A, 35B, 35C, etc.), with each of the recesses 30extending at a 45 degree angle relative to the axis of a given row 35,and with a recess 30 in one row 35 being aligned with an offset recess30 in an adjacent row 35. An inlet 40 is connected to a far side recess30 on the ultimate row 35A, and an outlet 45 is formed adjacent to theopposite far side recess 30 on the same ultimate row 35A. An inlet 50 isconnected to a far side recess 30 on the penultimate row 35B, and anoutlet 55 is formed adjacent to the opposite far side recess 30 on thesame penultimate row 35B. The antepenultimate row 35C lacks both aninlet and an outlet. An inlet 60 is connected to a far side recess 30 onthe ante-antepenultimate row 35D, and an outlet 65 is formed adjacent tothe opposite far side recess 30 on the same ante-antepenultimate row35D.

In one preferred form of the invention, and looking now at FIG. 9,recesses 30, inlets 40, 50, 60, and outlets 45, 55, 65 are all formed inthe bottom surface of top plate 20 using a conventional etching processof the sort well known in the etching arts. Preferably, recesses 30,inlets 40, 50, 60 and outlets 45, 55, 65 are etched in the bottomsurface of a glass plate. Alternatively, recesses 30, inlets 40, 50, 60and outlets 45, 55, 65 may be formed in a silicon plate, a plasticplate, a ceramic plate, a quartz plate, a metal oxide plate or otherappropriate substrate material.

Bottom plate 25 has a plurality of recesses 70 formed on its topsurface, with recesses 70 being arranged in a plurality of rows 75(i.e., 75A, 75B, 75C, etc.), with each of the recesses 70 extending at a45 degree angle relative to the axis of a given row 75, and with arecess 70 in one row 75 being aligned with an offset recess 70 in anadjacent row 75. An outlet 80 is connected to a far side recess 70 onthe ultimate row 75A. An outlet 85 is connected to a far side recess 70on the penultimate row 75B. The antepenultimate row 75C lacks an outlet.An outlet 90 is connected to a far side recess 70 on theante-antepenultimate row 75D. In addition, the plurality of bar channels10 are formed on the top surface of bottom plate 25, with each of thebar channels 10 being connected to a recess 70 in theante-ante-antepenultimate row 75E (see FIG. 8), and with each of the barchannels 10 extending parallel to one another and perpendicular to theaxis of rows 75.

In one preferred form of the invention, and looking now at FIG. 9,recesses 70, outlets 80, 85, 90, and bar channels 10 are all formed inthe top surface of bottom plate 25 using a conventional etching processof the sort well known in the etching arts. Preferably, recesses 70,outlets 80, 85, 90, and bar channels 10 are etched in the top surface ofa glass plate. Alternatively, recesses 70, outlets 80, 85, 90, and barchannels 10 may be formed in a silicon plate, a plastic plate, a ceramicplate, a quartz plate, a metal oxide plate or other appropriatesubstrate material.

Looking next at FIG. 10, top plate 20 is assembled on top of bottomplate 25 so that recesses 30 in top plate 20 communicate with recesses70 in bottom plate 25. More particularly, when top plate 20 is assembledon top of bottom plate 25 in this manner, recesses 30 in top plate 20will cooperate with recesses 70 in bottom plate 25 so as to initiallyform a plurality of continuous rows 95 (i.e., 95A, 95B, 95C, 95D, etc.)in multiplexed volumetric bar chart chip 5, with the inlet 40 ofultimate row 95A being connected with the outlet 45 of ultimate row 95A,with the inlet 50 of the penultimate row 95B being connected with theoutlet 55 of the penultimate row 95B, and with the inlet 60 of theante-antepenultimate row 95D being connected with the outlet 65 of theante-antepenultimate row 95D. As noted above, the antepenultimate row95C lacks both an inlet and an outlet.

Still looking now at FIG. 10, it will be appreciated that, due to thedispositions of recesses 30 in top plate 20 and recesses 70 in bottomplate 25, an oblique slide of top plate 20 relative to bottom plate 25disrupts the aforementioned rows 95 and causes them to transform into aplurality of continuous columns 100 (i.e., 100A, 100B, 100C, etc.), witheach column 100 being in fluid communication with one of theaforementioned bar columns 10.

In view of the foregoing construction, multiplexed volumetric bar chartchip 5 can be used to simultaneously determine the quantity of multipleproteins present in a sample, with the quantity of each specific proteinbeing indicated in a particular one of the plurality of bar channels 10.

More particularly, and referring now to FIGS. 11 and 12, and as willhereinafter be discussed in further detail below, during manufacture ofmultiplexed volumetric bar chart chip 5, a different protein-specificantibody is bonded in a recess 30 of the penultimate row 35B. As aresult, after the bottom plate 20 and top plate 25 are assembledtogether, row 75B will contain a series of different protein-specificantibodies, with a different protein-specific antibody being located ineach recess 30 of the row 75B.

Prior to use, hydrogen peroxide (H₂O₂) is introduced into inlet 40 ofmultiplexed volumetric bar chart chip 5, whereby to fill the ultimaterow 75A of multiplexed volumetric bar chart chip 5 with hydrogenperoxide. Red ink (or some other colored material which is readilydiscernible through top plate 25 and against bottom plate 20) isintroduced into inlet 60 of multiplexed volumetric bar chart chip 5,whereby to fill the ante-antepenultimate row 75D of multiplexedvolumetric bar chart chip 5 with red ink. Antepenultimate row 75C isintentionally left blank to serve as an air spacer, thereby avoidingdirect contact between a sample and the red ink.

Then, when a sample is to be checked for the presence and quantity ofspecific proteins (i.e., the proteins which will bind to theprotein-specific antibodies already bound to the recesses 30 of row75B), the sample is introduced into inlet 50 of multiplexed volumetricbar chart chip 5 so that the sample fills the penultimate row 75B. Thisaction causes the sample to mix with the different protein-specificantibodies which are bonded to bottom plate 20 in the recesses 30, sothat the target proteins bind to the appropriate protein-specificantibodies in the recesses 30. Significantly, each target protein bindsto only one protein-specific antibody, and such binding takes place inonly one of the recesses 30 in the penultimate row 75B. Thereafter, thepenultimate row 75B is flushed so as to remove any materials which arenot bound to a protein-specific antibody.

Next, catalase is introduced into inlet 50 of multiplexed volumetric barchart chip 5 so as to fill the penultimate row 75B. This action causesthe catalase to bind to the target proteins which are themselves boundto the protein-specific antibodies in the recesses 30. It will beappreciated that, to this end, the catalase is a mixture of all thecatalase detecting probes required for binding to the target proteins(e.g., silica nanoparticles conjugated with detecting antibodies andcatalase molecules). Then excess catalase is rinsed from the penultimaterow 75B.

Thereafter, top plate 25 is slid obliquely relative to bottom plate 20,causing rows 75 (i.e., 75A, 75B, 75C, 75D, etc.) to be disrupted andtransformed into columns 100 (i.e., 100A, 100B, 100C, etc.). As thisrow-to-column transformation occurs, each recess 30 (containing theprotein-specific antibodies and any target proteins bound thereto andany catalase bound thereto) previously located in penultimate row 75Bbecomes incorporated as a section of a specific column 100 (i.e., 100A,100B, 100C, etc.). In addition, as this row-to-column transformationoccurs, the hydrogen peroxide contained in row 75A is permitted toadvance up each of the columns 100 and thereby mix with any catalasebound to the target proteins (which are themselves bound to theprotein-specific antibodies), the mixing of which causes a reactionwhich releases oxygen gas. The oxygen gas is produced in proportion tothe quantity of catalase present in a given column (and hence inproportion to the quantity of target proteins which are present in agiven column). Thus, the quantity of oxygen gas produced in a givencolumn 100 is proportional to the quantity of target proteins which arepresent in a given column 100, with each of the columns 100 containing adifferent target protein (by virtue of the fact that each of the columns100 contains a different protein-specific antibody). The oxygen gasproduced by the reaction accumulates within the limited volume ofcolumns 100 and causes an increase in pressure, which propels the redink contained in columns 100 into and along bar columns 10, with the inkadvancing a distance along bar columns 10 which is proportional to thequantity of oxygen gas produced in that column, which is in turnproportional to the quantity of the target proteins which are bound tothe protein-specific antibodies disposed in the recesses associated withthat column.

As a result of the foregoing, by disposing different protein-specificantibodies in different ones of the recesses 30 of rows 35 of bottomplate 20, multiplexed volumetric bar chart chip 5 can be used tosimultaneously determine the quantity of multiple proteins present in asample, with the quantity of each protein being indicated in aparticular one of a plurality of bar channels 10. See, for example,FIGS. 13-16, where FIG. 13 shows multiplexed volumetric bar chart chip 5prior to the oblique sliding of top plate 25 relative to bottom plate20, and FIGS. 14-16 show the test results for various samples.

FIGS. 17-20 show specific steps in the foregoing process. Specifically,FIG. 17 shows a protein-specific antibody being bound in a recess 30 ofbottom plate 20; FIG. 18 shows a sample being loaded into a recess 30 ofbottom plate 20, whereby to bind a target protein to a protein-specificantibody; FIG. 19 shows catalase being loaded into a recess 30 so as tobind catalase to a target protein (which is itself bound to aprotein-specific antibody); and FIG. 20 shows hydrogen peroxide beingloaded into a recess 30, whereby to release oxygen gas in proportion tothe quantity of target protein present in a recess 30.

If desired, the same protein-specific antibody can be bound in multiplerecesses 30 of penultimate row 35B of bottom plate 20, whereby toprovide redundancy.

FIGS. 21-32 are a schematic series of views showing the assembly andoperation of the multiplexed volumetric bar chart chip in one preferredform of the present invention.

FIGS. 33-45 are a schematic series of views showing how, over time, theink in a given bar channel advances a distance along that bar channelwhich is proportional to the quantity of the target protein which arebound to the protein-specific antibody disposed in the recess associatedwith that bar channel, whereby to indicate, in multiplexed volumetricbar chart form, the results of a simultaneous multi-protein assay.

The novel method and apparatus of the present invention provides instantand visual quantitation of target biomarkers or other molecular analytesand provides a visualized bar chart without the use of instruments, dataprocessing or graphic plotting. Thus, since the novel method andapparatus of the present invention does not require the use of complexinstruments, the novel method and apparatus of the present invention canbe easily used as a point of care determination of the quantity of aprotein (and, preferably, the quantity of multiple proteins) present ina sample. More particularly, the novel method and apparatus of thepresent invention can be used as a point of care determination of thequantity of protein, nucleic acid, peptide, sugar, organic compounds,polymer, metal ions, and other molecular analytes, as well as thequantity of bacteria, cells, and particles.

In the foregoing description, gas is generated by the reaction of anELISA probe with a reagent, and specifically, gas is generated by thereaction of the ELISA probe (i.e., the protein-specific antibody whichis bound to the target protein which is bound to the catalase) withhydrogen peroxide. It is important to note that many other combinationsof a probe and a reagent may be used to generate gas. By way of examplebut not limitation, such probe and reagent combination may includecatalase and hydrogen peroxide, platinum film or particles and hydrogenperoxide, catalase and carbamide peroxide, zinc and chloric acid, ironand chloric acid, and other similar combinations. Thus, since themultiplexed volumetric bar chart chip readout is based on the volumetricmeasurement of a gas generation, many fast responsive gas generationschemes can be used for the system, including catalase with hydrogenperoxide, catalase and carbamide peroxide, zinc and chloric acid, ironand chloric acid, and other similar combinations.

Furthermore, the multiplexed volumetric bar chart chip is based on asandwich assay. In the foregoing description, a capture antibody bindsto an analyte and a detecting antibody conjugated with a catalase probeindicates the amount. Thus, the sandwich scheme is made up of captureantibody/analyte/detecting antibody conjugated with a catalase probe.

This type of sandwich scheme could also be extended to nucleic acidhybridization, where the sandwich is capture DNA strand/targetstrand/detecting DNA strand (i.e., the target strand has a first halfcomplimentary to the capture DNA strand and a second half complimentaryto the detecting DNA strand). By way of example but not limitation, seeFIG. 46, which shows specific steps that are performed in accordancewith a DNA assay scheme and oxygen generation mechanism.

Additionally, this type of sandwich scheme could also be extended tohydrogen bonding, electrostatic reaction or interaction, or covalentbonding, where the target analyte is captured by a surface with acoating that can adhere the analyte by either hydrogen bonding,electrostatic reaction or interaction or the formation of a covalentbond. The readout of the adhered or bonded analyte can then be detectedby the detecting antibody with a catalase probe. The sandwich of thesetypes are surfaces (with adhesion forces of hydrogen bonding,electrostatic interaction or covalent bonding)/analyte/probe ofdetecting antibody with catalase.

In another embodiment of the present invention, and looking now at FIG.47, a novel multiplexed volumetric bar chart chip 200 is provided whichmay be used in accordance with the present invention to determine thequantity of a target protein or other types of biomarkers or otheranalytes, wherein the signal for determining the quantity of the targetprotein or other types of biomarkers or other analytes is amplified.

More particularly, multiplexed volumetric bar chart chip 200 comprisestwo glass plates, a transparent top plate 220 and a bottom plate 225(which may or may not be transparent).

Top plate 220 and bottom plate 225 are similar to top plate 20 andbottom plate 25 discussed above, except that the plurality of rows arearranged on the multiplexed volumetric bar chart chip 200 so that therecesses in the rows are filled with the ELISA reagents (Assay) (i.e.,the protein-specific antibody, with the sample and catalase boundthereto), hydrogen peroxide, platinum film, hydrogen peroxide, platinumfilm, hydrogen peroxide, platinum film and ink.

As the ELISA reagent reacts with the hydrogen peroxide, oxygen isgenerated, with that oxygen being proportional to the quantity of thetarget antibody present in the sample. The oxygen generated by the ELISAreaction in turn drives a quantity of unreacted hydrogen peroxide (thatis proportional to the quantity of oxygen produced from the ELISAreaction) into the next row of the chip (which contains platinum film).When this unreacted hydrogen peroxide passes into the row containing theplatinum film, additional oxygen is generated, with the quantity ofoxygen generated being proportional to (but greater than) the quantityof oxygen produced from the original ELISA reaction). This processcascades down the successive rows of the chip and, with each step, theamount of oxygen produced is proportional to (but successively greaterthan) the original quantity of oxygen produced by the ELISA reaction,which is in turn proportional to the quantity of the target protein orother types of biomarkers or other analytes present in the sample.However, since more oxygen is produced by each successive hydrogenperoxide/platinum film reaction, the signal (i.e., the advancement ofthe red ink in the plurality of channels) is amplified. Since theadvancement of the red ink is the sum of the catalase reacting withhydrogen peroxide and the results of the platinum film reacting withhydrogen peroxide over three steps, multiplexed volumetric bar chartchip 200 exhibits a higher sensitivity than the multiplexed volumetricbar chart chip 5 discussed above. See, for example, FIG. 48, which showsimages of hydrogen peroxide solution being pushed into successiveplatinum wells. Due to the accumulated volume of oxygen at differentstages of the chip, more hydrogen peroxide was pushed into the platinumwells at the higher stage than at the lower stage.

In still another embodiment of the present invention, and looking now atFIGS. 49 and 50, a novel multiplexed volumetric bar chart chip 300 isprovided. Multiplexed volumetric bar chart chip 300 is similar tomultiplexed volumetric bar chart chip 5 discussed above, except thatmultiplexed volumetric bar chart chip 300 is manufactured so as toreduce the reagent loading and washing steps required for a user.

In this embodiment, the ELISA reagents (i.e., the washing buffer,catalase probe and washing buffer) can be preloaded in the multiplexedvolumetric bar chart chip during the manufacturing stage (e.g., at thelocations shown in FIG. 49). At the time of use, the sample ispositioned in the multiplexed volumetric bar chart chip (e.g., at thelocation shown in FIG. 49). Then, the multiplexed volumetric bar chartchip is slid vertically so that the sample, washing buffer, catalaseprobe and washing buffer are sequentially passed through the ELISAreagent row of the chip, whereby to prepare the ELISA row of the chip ina single action. Subsequently, the multiplexed volumetric bar chart chipcan be slid in the oblique direction so as to activate the oxygenreaction and generate the desired results.

In this form of the invention, the user will only need to load thesample into the chip and then slide the chip obliquely so as to activatethe assay process.

FIG. 51 shows another form of the present invention in which themultiplexed volumetric bar chart chip is configured to load the ELISArow of the chip through a horizontal motion.

Modifications Of The Preferred Embodiments

It should be understood that many additional changes in the details,materials, steps and arrangements of parts, which have been hereindescribed and illustrated in order to explain the nature of the presentinvention, may be made by those skilled in the art while still remainingwithin the principles and scope of the invention.

What is claimed is:
 1. Apparatus for determining the quantity of atarget protein and other types of biomarkers or analytes present in asample, the apparatus comprising: a top plate comprising a plurality ofrecesses arranged to form a plurality of rows extending parallel to oneanother; and a bottom plate comprising a plurality of recesses arrangedto form a plurality of rows extending parallel to one another, and aplurality of channels extending perpendicularly to the plurality of rowsof the bottom plate; wherein the top plate and the bottom plate areassembled together so that the top plate is on top of the bottom plateand the recesses of the top plate communicate with the recesses of thebottom plate so as to form a plurality of rows; and wherein at least oneof the top plate and the bottom plate is configured to slide relative tothe other of the top plate and the bottom plate in order to form aplurality of columns, with each of the plurality of columns incommunication with each of the plurality of channels.
 2. Apparatusaccording to claim 1 wherein the top plate is transparent.
 3. Apparatusaccording to claim 1 wherein at least one of the plurality of rowsformed in the top plate comprises an inlet and an outlet.
 4. Apparatusaccording to claim 1 wherein the recesses in the top plate and therecesses in the bottom plate extend at a 45 degree angle relative to theaxis of a row.
 5. Apparatus according to claim 1 wherein each of theplurality of channels is connected to a recess formed in the bottomplate.
 6. Apparatus according to claim 1 further comprising aprotein-specific antibody bound in at least one recess forming one ofthe plurality of rows of the top plate.
 7. Apparatus according to claim6 further comprising a sample positioned in the at least one recesscontaining the protein-specific antibody.
 8. Apparatus according toclaim 7 further comprising a catalase positioned in the at least onerecess containing the protein-specific antibody and the sample. 9.Apparatus according to claim 6 further comprising a plurality ofprotein-specific antibodies each bound in a separate recess forming oneof the plurality of rows of the top plate.
 10. Apparatus according toclaim 9 further comprising a sample positioned in each recess containinga protein-specific antibody.
 11. Apparatus according to claim 6 furthercomprising hydrogen peroxide positioned in a recess in a row adjacent tothe row containing the protein-specific antibody.
 12. Apparatusaccording to claim 6 further comprising ink positioned in a recess in arow adjacent to the plurality of channels.
 13. Apparatus according toclaim 1 further comprising a bar code reader for detecting thelongitudinal position of ink contained in the plurality of channels. 14.A method for determining the quantity of a target protein and othertypes of biomarkers or analytes present in a sample, the methodcomprising: providing apparatus comprising: a top plate comprising aplurality of recesses arranged to form a plurality of rows extendingparallel to one another; and a bottom plate comprising a plurality ofrecesses arranged to form a plurality of rows extending parallel to oneanother, and a plurality of channels extending perpendicularly to theplurality of rows of the bottom plate; wherein the top plate and thebottom plate are assembled together so that the top plate is on top ofthe bottom plate and the recesses of the top plate communicate with therecesses of the bottom plate so as to form a plurality of rows; andwherein at least one of the top plate and the bottom plate is configuredto slide relative to the other of the top plate and the bottom plate inorder to form a plurality of columns, with each of the plurality ofcolumns in communication with each of the plurality of channels; bindinga protein-specific antibody in at least one recess forming one of theplurality of rows of the top plate; positioning hydrogen peroxide in arecess adjacent to the row containing the protein-specific antibody;positioning ink in a recess in a row adjacent to the plurality ofchannels; positioning a sample in the at least one recess containing theprotein-specific antibody; positioning a catalase in the at least onerecess containing the protein-specific antibody and the sample; slidingone of the top plate and the bottom plate relative to the other of thetop plate and the bottom plate so as to form the plurality of columns,with each column being in communication with one of the plurality ofchannels; and determining the quantity of the target protein and otherbiomarker and other molecular analyte present in the sample by detectingthe longitudinal position of the ink contained in the plurality ofchannels.
 15. A method according to claim 14 wherein the top plate istransparent.
 16. A method according to claim 14 wherein at least one ofthe plurality of rows formed in the top plate comprises an inlet and anoutlet.
 17. A method according to claim 14 wherein the recesses in thetop plate and the recesses in the bottom plate extend at a 45 degreeangle relative to the axis of a row.
 18. A method according to claim 14wherein each of the plurality of channels is connected to a recessformed in the bottom plate.
 19. A method according to claim 14 furthercomprising a plurality of protein-specific antibodies each bound in aseparate recess forming one of the plurality of rows of the top plate.20. A method according to claim 14 further comprising using a bar codereader to detect the longitudinal position of ink contained in theplurality of channels.
 21. A method for determining the quantity of atarget analyte present in a sample, the method comprising: providingapparatus comprising: a top plate comprising a plurality of recessesarranged to form a plurality of rows extending parallel to one another;and a bottom plate comprising a plurality of recesses arranged to form aplurality of rows extending parallel to one another, and a plurality ofchannels extending perpendicularly to the plurality of rows of thebottom plate; wherein the top plate and the bottom plate are assembledtogether so that the top plate is on top of the bottom plate and therecesses of the top plate communicate with the recesses of the bottomplate so as to form a plurality of rows; and wherein at least one of thetop plate and the bottom plate is configured to slide relative to theother of the top plate and the bottom plate in order to form a pluralityof columns, with each of the plurality of columns in communication witheach of the plurality of channels; binding a capture agent in at leastone recess forming one of the plurality of rows of the top plate,introducing a sample into the at least one recess so that an analytecontained in the sample is bound to the capture agent, and binding aprobe to the bound analyte; and positioning a reagent in a recessadjacent to the row containing the capture agent, bound analyte andbound probe; and positioning ink in a recess in a row adjacent to theplurality of channels; sliding one of the top plate and the bottom platerelative to the other of the top plate and the bottom plate so as toform the plurality of columns, with each column being in communicationwith one of the plurality of channels; and determining the quantity ofthe analyte present in the sample by detecting the longitudinal positionof the ink contained in the plurality of channels.
 22. A methodaccording to claim 21 wherein the probe and reagent are selected fromthe group consisting of catalase and hydrogen peroxide, platinum film orparticles and hydrogen peroxide, catalase and carbamide peroxide, zincand chloric acid and iron and chloric acid.
 23. Apparatus according toclaim 1 further comprising an ELISA probe to react with a reagent togenerate gas.
 24. Apparatus according to claim 23 wherein the ELISAprobe and reagent are selected from the group consisting of catalase andhydrogen peroxide, platinum film or particles and hydrogen peroxide,catalase and carbamide peroxide, zinc and chloric acid and iron andchloric acid.
 25. A method according to claim 14 wherein advancement ofink contained in the plurality of channels indicates the quantity of thetarget protein or other biomarker or other molecular analyte present inthe sample.
 26. Apparatus according to claim 1 wherein advancement ofink contained in the plurality of channels indicates the quantity of thetarget protein or other biomarker or other molecular analyte present inthe sample.
 27. Method according to claim 21 wherein advancement of inkcontained in the plurality of channels indicates the quantity of theanalyte present in the sample.
 28. Apparatus according to claim 1wherein the top plate and the bottom plate are made from a materialselected from the group consisting of glass, silicon, plastics,ceramics, quartz and metal oxide.
 29. Method according to claim 14wherein the top plate and the bottom plate are made from a materialselected from the group consisting of glass, silicon, plastics,ceramics, quartz and metal oxide.
 30. Method according to claim 21wherein the top plate and the bottom plate are made from a materialselected from the group consisting of glass, silicon, plastics,ceramics, quartz and metal oxide.
 31. Apparatus according to claim 1wherein the plurality of columns in communication with the plurality ofchannels form a readout panel for determining the quantity of the targetprotein and other types of biomarkers or analytes present in the sample.32. A method according to claim 14 wherein the plurality of columns incommunication with the plurality of channels form a readout panel fordetermining the quantity of the target protein and other types ofbiomarkers or analytes present in the sample.
 33. A method according toclaim 21 wherein the plurality of columns in communication with theplurality of channels form a readout panel for determining the quantityof the analyte present in the sample.
 34. A method according to claim 21wherein the bound analyte and probe form an assay sandwich, and theassay sandwich is formed by one selected from the group consisting ofELISA, nucleic acid hybridization, hydrogen bonding, electrostaticreaction and formation of covalent bond.
 35. Apparatus according toclaim 1 further comprising an assay and a probe bound in at least onerecess forming one of the plurality of rows of the top plate. 36.Apparatus according to claim 35 wherein the assay and the probe form anassay sandwich, and the assay sandwich is formed by one selected fromthe group consisting of ELISA, nucleic acid hybridization, hydrogenbonding, electrostatic reaction and formation of covalent bond. 37.Apparatus according to claim 1 wherein the target protein, biomarker oranalyte present in a sample is selected from the group consisting of aprotein, a nucleic acid, a peptide, a sugar, an organic compound, apolymer, a metal ion, bacteria, cells and particles.
 38. A methodaccording to claim 14 wherein the target protein, biomarker or analytepresent in a sample is selected from the group consisting of a protein,a nucleic acid, a peptide, a sugar, an organic compound, a polymer, ametal ion, bacteria, cells and particles.
 39. A method according toclaim 21 wherein the target analyte present in a sample is selected fromthe group consisting of a protein, a nucleic acid, a peptide, a sugar,an organic compound, a polymer, a metal ion, bacteria, cells andparticles.
 40. Apparatus according to claim 1 further comprising: acapture agent bound in at least one recess forming one of the pluralityof rows of the top plate, a sample comprising at least one analyte, theanalyte being bound to the capture agent, and a probe bound to the boundanalyte; a reagent positioned in at least one recess in a row adjacentto the row containing the capture agent, the bound analyte and the boundprobe, wherein reaction of the reagent with the probe produces a gas ina quantity reflective of the quantity of the analyte present in thesample; and ink positioned in a row adjacent to the plurality ofchannels.
 41. Apparatus according to claim 40 wherein the reagent ispositioned in a plurality of rows, with one row being adjacent to therow containing the capture agent, the bound analyte and the bound probe,and with the remainder of the rows containing the reagent alternatingwith rows containing an amplifier agent, wherein reaction of the reagentwith the amplifier agent produces a gas in a quantity larger than, butproportional to, the quantity of gas produced by the reaction of thereagent with the probe.
 42. A method according to claim 21 whereinreaction of the reagent with the probe produces a gas in a quantityreflective of the quantity of the analyte present in the sample, andfurther wherein the reagent is positioned in a plurality of rows, withone row being adjacent to the row containing the capture agent, thebound analyte and the bound probe, and with the remainder of the rowscontaining the reagent alternating with rows containing an amplifieragent, wherein reaction of the reagent with the amplifier agent producesa gas in a quantity larger than, but proportional to, the quantity ofgas produced by the reaction of the reagent with the probe. 43.Apparatus according to claim 1 further comprising a capture agent boundin at least one recess forming one of the plurality of rows of the topplate.
 44. Apparatus according to claim 43 further comprising a samplecomprising at least one analyte and a probe.